Herpes simplex virus type 1 (HSV-1) is ubiquitous in the human population and causes a variety of clinically significant acute diseases which can be life-threatening to immunocompromised individuals. HSV-1 also establishes life-long latent infections characterized by periodic reactivation, virus shedding and recurrent disease. The ability of the virus to establish latent infections which cannot be cured by the immune response or existing antiviral drugs is the reason that HSV-1 continues to be a significant human pathogen. In this proposal, three senior herpesvirologists will conduct a series of collaborative studies designed to elucidate the mechanism of establishment, maintenance and reactivation of HSV-1 latency by identifying the viral and cellular proteins and activities that result in latency and reactivation. Project 1 will focus on the state of viral chromatin during latent infection and will investigate the roles of ICPO and the latency-associated transcripts (LATs) in regulating the chromatization of latent genomes in collaboration with Projects 2 and 3. Project 1 will also examine the long-term immune response to HSV-1 and HSV-1 latent infection by defining the role of TLR2 signaling in the immune response. Project 2 will investigate the molecular and genetic mechanism by which clinical HSV-1 isolates become drug-resistant while retaining pathogenesis and permitting reactivation. In collaboration with Projects 1 and 3, Project 2 will also focus on how late gene expression is repressed by interferon gamma (IFN-y) and the LATs and later reactivated. Also in collaboration with Projects 1 and 3, Project 2 will examine the effects of IFN-y and newly discovered viral miRNAs on host gene expression. Project 3, in collaboration with Projects 1 and 2, will focus exclusively on the mechanisms of HSV-1, reactivation by attempting to identify the cellular proteins that mediate stress-induced reactivation, identifying the promoter elements in viral genes that respond to stress, and determining the effects of cdks on the activities of viral proteins induced by stress. Collectively, these studies will provide new insight into the mechanisms of HSV-1 latency and reactivation and define novel approaches to intervention in the HSV-1 life-cycle. PROJECT 1 PI: David M. Knipe Title: Gene Regulatory &Immunologic Mechanisms in Herpes Latency Description (provided by applicant): Latent infection by herpes simplex virus provides a means for persistence of the virus in its human host and for evasion of the host immune response, which attempts to clear the virus. The long-term goals of this research are to define the mechanisms of latent infection by herpes simplex virus and the host immune response to acute and latent infection. In this proposed study we will study the mechanisms of regulation of viral gene expression by histone modifications, the roles of the viral LAT and ICPO genes in the chromatin state of the viral latent chromosome, and the mechanism of long-term immune responses to HSV antigens. Our specific aims in Project 1 are: 1. To define the state of chromatin on lytic genes and the LAT gene during latent infection by defining the forms and posttranslational state of histones associated with HSV lytic genes and LAT gene using chromatin immunoprecipitation (ChIP), by testing for the presence of heterochromatin proteins on viral genes by ChIP analysis, by defining the chromatin distribution across several representative immediate and early viral genes as an initial stage of defining the "epigenome" of latent herpes simplex virus, by defining the histone modifications on additional HSV genes, and by determining if methylation of H3 K4 (active chromatin) and K9 (heterochromatin) are independent or mutually exclusive histone H3 modifications on individual HSV lytic-gene promoters. 2. To define the role of LAT and ICPO in regulation of chromatin structure on latent viral genomes in collaboration with projects 2 and 3 by studying the chromatin structure of additional LAT gene mutant viruses, by determining if the LAT gene effects on chromatin are due to RNA silencing or X inactivation mechanisms, by determining if the HSV immediate early ICPO protein affects viral chromatin structure during latent infection, and by determining the phenotype of a LAT/ICPO double mutant virus. 3. To define the role of TLR2 signaling in long-term immune responses to HSV and in HSV latent infection by examining in collaboration with projects 2 and 3: The role of TLR2 in persistent expression of chemokines and cytokines in HSV-infected mice, the role of TLR2 in long-term humoral responses, the role of TLR2 in long-term HSV-specific CD8+ T cell infiltrates in ganglia, and the role of TLR2 signaling in latent infection. We are beginning to understand some of the mechanisms of latent infection by herpes simplex virus, and the proposed studies should provide further insight into the role of viral gene products and the host immune response in HSV latent infection. These studies may ultimately lead to new approaches to interrupt latent infection by herpes simplex virus.